Mycoplasmas are the smallest free-living microorganisms known to man. They are smaller than bacteria and larger than most viruses, measuring about 10.times.200 nm in size. Because they lack a rigid cell wall, are very pleomorphic and stain poorly with dyes, the individual organisms are very difficult to recognize with light microscopy. In fact, the species Mycoplasma pneumoniae (M. pneumoniae), a pathogen causing human primary atypical pneumonia and upper respiratory tract infections, was initially given such ill-defined names as Eaton agent and pleuropneumonia-like organism (PPLO). Cultivation of the organism in cell-free media was not achieved until early 1960s. Methods for isolation, propagation and specific identification of M. pneumoniae from patients' excretions remain highly technical and are performed routinely only in a few laboratories. Most clinical laboratories depend on either nonspecific serologic tests, such as cold agglutinin and MG streptococcus tests, or the complement fixation test for establishing diagnosis of M. pneumoniae infections in humans. [Textbook of Medicine, by P. B. Beeson and W. McDermott, W. B. Saunders Co., Philadelphia, 1975, pp. 270-274. Medical Microbiology and Infectious Diseases, by A. I. Braude, W. B. Saunders Co., Philadelphia, 1981, pp. 925-929. Respiratory Infections: Diagnosis and Management, by J. E. Pennington, Raven Press, New York, 1983, pp. 251-257.]
Several investigators have attempted to use an indirect fluorescent antibody technique, using M. pneumoniae grown in solid or liquid media as antigens. In 1962 Chanock, Hayflick and Barile reported successful transferring of mycoplasmal colonies from solid agar media to glass slides and demonstrating specific antibodies against these colonies in convalescent serum samples of patients recovering from M. pneumoniae infection. [Chanock, R. M., Hayflick, L. and Barile, M. F.; Proc. Nat. Acad. Science, 48:41-49, 1962.] However, because the colonies of the microorganisms are largely embedded in the agar and not readily removed by mechanical scraping, the researchers had to place the agar medium with grown colonies on the slide, then melted the agar away by raising its temperature to at least 80.degree. to 85.degree. C. in order to remove the agar. Unfortunately, by doing so the antigen is partially denatured, and the technique cannot be used to produce a large number of slides with adherent colonies for diagnostic purpose in a clinical laboratory.
In 1978, Silis and Andrews suggested using the granular deposits from a liquid culture of M. pneumoniae as antigen for the indirect fluorescent antibody test instead of colonies grown in a solid medium so that the heating procedure can be omitted. These granular deposits were presumably mycoplasmal colonies. [Silis, M. and Andrews, B. E.: A simple test for Mycoplasma pneumoniae IgM. Zentralblatt fur Bakteriologie, I. Abteilung Original, 241:239-240, 1978.] However, this approach has not been widely used because antigenic specificity of these deposits or whole colonies has not been proven. In fact, there is significant antigenic cross reaction between M. pneumoniae and M. genitalium colonies. [Lind, K., Lindhardt, B., Schutten, H. J., Blom, J. and Christiansen, C.: Serological Cross-Reactions between Mycoplasma Genitalium and Mycoplasma Pneumoniae, J. Clinical Microbiology 20: 1036-1043, 1984.]
Another approach to procure M. pneumoniae antigen for indirect fluorescent antibody test in a clinical laboratory is to use a commercial M. pneumoniae preparation which has been grown in a liquid medium, centrifuged and freeze-dried, and marketed by Wellcome Laboratories, Research Triangle Park, N.C., U.S.C., as reported by Carter and Carter. [Carter, J. B. and Carter, S. L.: Acute-Phase, Indirect Fluorescent Antibody Procedure for Diagnosis of Mycoplasma Pneumoniae Infection. Annals of Clinical and Laboratory Science, 13:150-155, 1983.] Unfortunately, by using this material as antigen, the authors reported that "a positive result appears as an applegreen, fluorescent slurry of particulate matter consistent with the almost submicroscopic morphology of the M. pneumoniae organism" (p. 152). In other words, the authors found it difficult to distinguish M. pneumoniae organisms from other particulate matters from the liquid media with confidence. It has been well-recognized by workers in the field of mycoplasma research that precipitates other than mycoplasma organisms invariably form in the agitated liquid media during incubation. The precipitates are not readily distinguishable from the highly pleomorphic minute microorganisms. Serum samples containing high titer of non-specific immunoglobulin M such as rheumatoid factors, may give rise to false positive results. [MP-1-IgM. Zeus.RTM. Technologies, Inc., Series No. 17000 M. 1984 (see "Limitations", No. 6), P. O. Box 177, Raritan, N.J. 08869.]
From these earlier publications by others, it has become clear that the only reliable morphologic criterion for recognizing the M. pneumoniae organisms is by its colony-forming characteristic, i.e., to work with concentrated, purified microcolonies. However, the whole colonies may contain non-specific antigens. Therefore, one must use colonies with abundant species-specific antigens which can be distinguished morphologically from non-specific antigens.
It is an object of the present invention to provide a method for selective propagation of colony-forming mycoplasma in dialysed, particle-free liquid media by "weeding out" the single growing units through a series of subcultures.
It is another object of the invention to provide a method for making an inoculum by breaking up of the tuberous floating colonies of mycoplasma by gentle homogenization into small fragments, but not into single growing units. The inoculum is used for the last culture in which each fragment grows into a medusoid in 4-5 days.
Another object of the invention is the identification of specific membrane antigen.
It is a further object of the invention to use aldehydes, desirably glutaraldehyde to stabilize the specific membrane antigens of mycoplasma.